Project description:Metastatic breast cancer cells disseminate to organs with a soft microenvironment. Whether and how tissue mechanical properties influence their response to treatment remains unclear. Here we found that a soft ECM empowers redox homeostasis. Cells cultured on a soft ECM display increased peri-mitochondrial F-actin promoted by Spire1C and Arp2/3 nucleation factors, and increased DRP1- and MIEF1/2-dependent mitochondrial fission. Changes in mitochondrial dynamics lead to increased mtROS production and activate the NRF2 antioxidant transcriptional response, including increased cystine uptake and glutathione metabolism. This retrograde response endows cells with resistance to oxidative stress and ROS-dependent chemotherapy drugs. This is relevant in a mouse model of metastatic breast cancer cells dormant in the lung soft tissue, where inhibition of DRP1 and NRF2 restored cisplatin sensitivity and prevented disseminated cancer cell awakening. We propose that targeting this mitochondrial dynamics- and redox-based mechanotransduction pathway could open new avenues to prevent metastatic relapse.
Project description:Hippocampal rat neurons have been cultured on very soft (100 Pa) and stiff (10 kPa) hydrogels for 7 days. On DIV7, the RNA has been extracted and sequenced. The goal of this experiment is to understand why neurons mature more quickly on soft gels compared to stiff gels.
Project description:Purpose: To identify genes and the molecular pathways involved in the MSCs response to extracellular matrix stiffness, we performed RNA-sequencing of MSCs which cultured in soft (2 kPa) and stiff (18 kPa) SA hydrogels. Methods: mRNA profiles of MSCs cultured in soft (2 kPa) and stiff (18 kPa) SA hydrogel for 48 h were generated by deep sequencing, in quadruplicate, using Illumina HiSeq 2000. Results: Using an optimized data analysis workflow, we identified 33950 transcripts in MSCs with BWA workflow. Conclusions: Our results present the detailed analysis of MSCs transcriptomes cultured in soft (2 kPa) and stiff (18 kPa) matrix, and found that matrix stiffness dominated multiple mRNA pathways in MSCs.
Project description:We report the expression profiles of MCF10A cells encapsulated in hydrogels of varying stiffness and composition. Cells were encapsulated for 7 days in either 1.) soft alginate and reconstituted basement membrane (rBM), 2.) stiff alginate and rBM, 3,) soft col-1 and rBM, or 4.) stiff col-1. We find global gene expression changes in response to enhanced ECM stiffness, independent of expression changes in response to col-1 exposure. These results provide a comprehensive study of the gene expression changes associated with increased ECM stiffness in addition to the gene expression changes associated with increased col-1 concentration in combination with, and independent of, ECM stiffness.
Project description:Dysregulation of vascular stiffness and cellular metabolism occur early in pulmonary hypertension (PH). Yet, the mechanisms by which biophysical properties of extracellular matrix relate to metabolic processes and downstream PH phenotypes remain undefined. In cultured endothelial and smooth muscle cells and confirmed in PH-diseased human samples, we found that ECM stiffening activates the mechanosensitive factors YAP/TAZ to increase glycolysis and induce glutaminase (GLS) expression and glutaminolysis. Glutaminolysis replenishes aspartate for anabolic biosynthesis, thus sustaining proliferation and migration within stiff ECM. In vitro GLS inhibition blocks aspartate production, consequently reprogramming entire cellular proliferative pathways, while aspartate restores proliferation. In a rat model in vivo, GLS inhibition prevents hemodynamic and histologic manifestations of PH. Thus, mechanical ECM stiffening sustains vascular cell growth and migration through YAP/TAZ-dependent glutaminolysis â a paradigm that advances our understanding of the connections of mechanical stimuli with dysregulated vascular metabolism and identifies new metabolic drug targets in PH. We used microarrays to decipher the global program of gene expression involved in response to matrix stiffening and determined the implication of glutaminolysis (GLS) in these process PAECs were transfected with an siRNA control (siNC) or a siRNA against GLS (siGLS) and cultivated on soft hydrogel (1kPa) or stiff hydrogel (50kPa). After 48h of transfection cells were lysate and RNA extract for hybridization on Affymetrix microarrays.
Project description:Primary human hepatic stellate cells (HSCs) isolated from healthy patients were purchased from Sciencell. They were seeded on either 400 Pa or 25000 Pa polyacrylamide hydrogels for 2 days and collected for RNA isolation. The goal of this study is to determine genes transcriptionally regulated by a stiff matrix. 2 RNA samples of HSCs on 400 Pa and 3 RNA samples of HSCs on 25600 Pa were sent to the University of Minnesota Genomics Center for RNA sequencing.
Project description:Transcriptional profiling of mouse chondrocytes cells comparing control untreated chondrocytes cells with chondrocytes cells plated in stiff and soft ECM. Goal was to determine the effects of ECM stiffness on gene expression.
Project description:Understanding how cells respond to the mechanics of their environment, and what affect senescence may have on this response, is important to gain a better understanding of mechanobiology, both in health and ageing-associated pathology. This experiment assessed the mRNA levels in early and late passage donor-matched human mesenchymal stem cells (MSCs) cultured for four days on soft (2 kPa) or stiff (25 kPa) collagen-I coated polyacrylamide (PA) gels. A minimum of three donors were analysed under each condition. Protein coding RNAs were sequenced with Illumina HiSeq technology. In a parallel experiment, protein was quantified by mass spectrometry proteomics.
Project description:Global transcriptome analysis showed that human lymphatic endothelial cells (LECs) grown on a soft matrix exhibit increased GATA2 expression, concomitant with a GATA2-dependent upregulation of genes involved in cell migration and lymphangiogenesis, including the key lymphangiogenic growth factor receptor VEGFR3. Affymetrix GeneChip analysis revealed regulation of 2771 transcripts above or below a 1.4-fold change (log2 fold change >0.5 or <-0.5) threshold on soft versus stiff matrices. Moreover, 406 (27%) of the 1485 transcripts that were increased and 207 (16 %) of the 1286 transcripts that were decreased on soft matrix were regulated in a GATA2 dependent manner.